Abstract Sea urchins have been valuable research models for more than a century, significantly contributing to our knowledge of fundamental biological processes like cell cycle regulation and developmental gene regulatory networks. While an increasing number of functional genomic resources are being developed for sea urchins, in vitro cell culture tools and methods for scalable transgenesis have remained elusive. Here, we successfully established embryonic cell lines from sea urchins (Lytechinus variegatus and Strongylocentrotus purpuratus) which recapitulated aspects of the developmental program in vitro, generating 3D structures and a variety of morphologically and functionally distinct cell types representing all three germ layers. Single-cell transcriptional profiles revealed signatures of stem cells and distinct cell clusters expressing markers of neurons, myocytes, skeletogenic, endodermal, and pigment cells. The ability to genetically manipulate cultured cells was demonstrated using a lentiviral vector, providing a scalable approach for mechanistic studies that significantly expand the utility of this model organism.